As the second leading cause of death and disability, ischemic stroke kills and disables millions of people each year. Tissue plasminogen activator (TPA), the only approved treatment, dissolves the culprit fibrin thrombus to restore blood flow and relieve the brain from ischemia. Unfortunately, after prolonged ischemia, TPA may cause serious or fatal complications; this restricts TPA use to a minority of stroke patients. Although TPA has provided a model for therapeutic fibrinolysis, recent data suggest a new paradigm that assigns a central role to regulatory molecules such as alpha-2-antiplasmin (a2AP) in determining outcomes after ischemic stroke. Clinical observations suggest that high a2AP levels may increase the risk of ischemic stroke and of TPA failure. In experiments that challenge the current therapeutic paradigm for fibrinolytic treatment of stroke, we have shown that a2AP markedly increases brain injury, in a dose-dependent fashion. Conversely, a2AP deficiency or monoclonal antibody inactivation of a2AP, profoundly reduces brain injury, apoptosis, hemorrhage, and swelling. Even after prolonged brain ischemia, a2AP inactivation reduces microvascular thrombosis and MMP-9 expression (a marker of acute inflammation). As a result, a2AP inactivation prevents death and disability after ischemic stroke. Thus, when compared to TPA, a2AP-inactivation appears to provide a safe and effective approach for improving stroke treatment and, through a NINDS collaboration, we are pursuing the development of a2AP inactivation therapy. This proposal seeks to determine the pathophysiologic mechanisms through which a2AP enhances ischemic brain injury after thromboembolic stroke. The organizing hypothesis is that a2AP acts through plasminogen-dependent mechanisms to enhance the development of microvascular thrombosis and impair downstream, microvascular perfusion. Through these mechanisms, a2AP promotes the development of inflammatory responses such as MMP-9 expression and neutrophil recruitment, which have acute deleterious effects. Aim 1 will test the hypothesis that a2AP's deleterious effects in ischemic stroke are due to diminished plasmin(ogen)-dependent, endogenous fibrinolysis that impairs microvascular blood flow through its effect on the culprit thrombus and the development of downstream, thrombin-dependent, microvascular thrombosis. We also propose to examine the hypothesis (Aim 2) that a2AP regulates the endogenous fibrinolytic system to affect the development of ischemic injury, hemorrhage, swelling and survival in thromboembolic stroke through inflammation-linked pathways that require MMP-9 activity and neutrophil deposition. Finally, we will use molecular complementation with specific a2AP mutants to examine the hypothesis that specific structural elements in the a2AP molecule selectively enhance adverse outcomes (such as neuronal injury, hemorrhage, etc.) in ischemic stroke.